N-benzoyl-5'-O-[bis(4-methoxyphenyl)phenylmethyl]-2'-deoxyadenosine

Endpoint summary

Description of key information

The potential of N-Benzoyl-5'-O-[bis(4-methoxyphenyl)phenylmethyl]-2'-deoxyadenosine (100% purity) to induce skin irritation (OECD 439) and eye irritation (OECD 492, OECD 437) was tested in suitable in vitro test methods. Based on the results, the target substance can be considered non-irritant to the skin. By assessing the results from both in vitro eye irritation tests in a weight-of-evidence approach, the target substance must be considered as irritating to the eye (Eye Irrit. 2, H319).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-23 to 2018-01-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 28th July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Firstly, 25 µL of sterile DPBS were applied to the epidermal surface in order to improve the contact between the powder and the epidermis. Afterwards, 25 mg (39 mg/cm²) of the test item were applied directly atop the EpiDerm tissue using an application spoon avoiding compression of the test item. The test item was spread to match size of the tissue by using a bulb-headed Pasteur pipette.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EpiDerm™-Standard Model (EPI-200-SIT, MatTek)
- Tissue batch number(s): 25850

EpiDerm Kit:
The EpiDerm tissues were provided as kits (e.g. EPI-200-SIT, MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm²); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport (Lot No.: 25849)
2x 24-well plates
8x 6-well plates
1x bottle of assay medium (DMEM-based medium, Lot No.: 101917TMB)
1x bottle of DPBS Rinse Solution (Lot No.: 062717MKGKA)
1x 1 vial 5% SDS Solution (TC-SDS-5%)
25 pieces Nylon Mesh circles (8 mm diameter, 200 µm pore)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for the first 35 +/- 1 min, afterwards the plates were placed under the sterile flow until 60 +/- 1 min incubation time of the first dosed tissue was over.
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 5 min
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the tissue viability after exposure and post-incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after exposure and post-incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg + 25 µL DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 30 µL DPBS (DPBS; Gibco, Cat. No. 14040-091, Lot No.: 1838067)

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 30 µL 5% SDS solution (TC-SDS-5%; MatTek, CAS No.: 151-21-3, Lot No.: 031617MGKA).

Duration of treatment / exposure:

60 min +/ 1 min

Duration of post-treatment incubation (if applicable):

42 h post-incubation

Number of replicates:

3 tissues per dose group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three tissues

Value:

106.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

For detailed results see Table 1 in box "Any other information on results incl tables" .

Results of the  Pre-Experiments:

The mixture of the 25 mg of the test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT was determined to be 0%.

The mixture of 25 mg of the test item per 300 µL aqua dest. or per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC was determined to be 0%.

Results of the main experiment:

Table 1: Result of the Test Item N-Benzoyl-5’-O-[bis(4-methoxyphenyl)phenylmethyl]-2’-deoxyadenosine

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.625	1.601	1.495	0.114	0.084	0.088	1.795	1.611	1.629
	1.644	1.592	1.528	0.113	0.084	0.088	1.794	1.594	1.644
OD570(Blank Corrected)	1.581	1.557	1.451	0.070	0.040	0.044	1.751	1.567	1.585
	1.600	1.548	1.484	0.069	0.040	0.044	1.750	1.550	1.600
Mean OD570of the Duplicates (Blank Corrected)	1.591	1.552	1.467	0.070	0.040	0.044	1.751	1.559	1.593
Total Mean OD570of 3 Replicate Tissues (Blank Corrected)	1.537*			0.051			1.634
SD OD570	0.063			0.016			0.102
Relative Tissue Viability [%]	103.5	101.0	95.5	4.5	2.6	2.9	113.9	101.4	103.6
Mean Relative Tissue Viability [%]	100.0			3.3**			106.3
SD Tissue Viability [%]***	4.1			1.0			6.7
CV [% Viabilities]	4.1			31.3			6.3

*Blank-corrected mean OD_{570 nm}of the negative control corresponds to 100% absolute tissueviability.

*Mean relative tissue viability of the three positive control tissues is≤ 20%.

***Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.

Table 2: Quality Criteria

	Value	Cut off	pass/fail
Mean Absolute OD570 nmNK	1.581	0.8 ≤ NK ≤ 2.8	pass
Relative Viability [%] PC	3.3	≤ 20%	pass
SD Viability[%]	1.0 – 6.7	≤ 18%	pass

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions, N-Benzoyl-5’-O-[bis(4-methoxyphenyl)phenylmethyl]-2’-deoxyadenosine showed no irritant effects. The relative mean tissue viability after 60 min of exposure and 42 h post-incubation was > 50%. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

In a primary dermal irritation study conducted according to OECD guideline 439, the EpiDerm™-Model (EPI-200-SIT) was topically exposed to N-Benzoyl-5’-O-[bis(4-methoxyphenyl)phenylmethyl]-2’-deoxyadenosine (100% purity) for 60 min followed by a 42 h post-incubation period. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS. The mean relative tissue viability (% negative control) was > 50% (106.3 %). Based on this result, the test item is classified as a non-irritant according to the UN GHS.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-11-22 to 2018-02-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted 28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method: This test uses the three-dimensional RhCE EpiOcular™ (MatTek). It consists of normal, human-derived epidermal keratinocytes and mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The MatTek EpiOcular™ model has been widely used as a research and testing model for many years.

- Description of the cell system used: The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium that is morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg

Duration of treatment / exposure:

6 ± 0.25 h

Observation period (in vivo):

n.a.

Duration of post- treatment incubation (in vitro):

Post exposure post-soak plate: 25 ± 2 min at room temperature
Post exposure post-treatment plate: 18 ± 0.25 h at 37 ± 1 °C

Number of animals or in vitro replicates:

2 tissues per dose group

Details on study design:

- Details of the test procedure used:
Upon receipt of the EpiOcular™, the tissues were equilibrated in the 24-well shipment plate to room temperature for about 15 min. The EpiOcular™ tissues were then transferred into 6-well plates containing 1 mL pre-warmed assay medium per well and incubated for 1 h at 37 +/- 1 °C, 5.0% CO2 / 95% air. Then the tissues were transferred into new 6-well plates containing 1 mL fresh assay medium per well and pre-incubated in a humidified incubator at 37 +/- 1 °C, 5.0% CO2 / 95% air for 16 - 24 h.
After the overnight incubation the tissues were pre-treated with 20 µL of DPBS and incubated for 30 ± 2 min in a humidified incubator at 37 +/- 1 °C, 5.0% CO2 / 95% air to mimic the wet conditions of the human eye. Afterwards, the tissues were treated with each dose group in duplicate, starting with the negative and positive control. The test item was applied on the tissues placed on a sterile surface. Start time was recorded with dosing of the first tissue and occurred sequentially for the other tissues, e.g. in one-minute intervals. After dosing, the tissues were placed back into the culture medium. Then the 6-well plate(s) were incubated for 6 ± 0.25 h at 37 +/- 1 °C, 5.0% CO2 / 95% air until the 6 ± 0.25 h of the first dosed tissue was over. At the end of the exposure period, the test item and control substances were removed by extensively rinsing the tissue with DPBS, occurring also in sequential order, e.g. in one-minute intervals. Excess DPBS was removed by decanting the insert and blotting the bottom with a blotting paper. After rinsing, the tissues were transferred to and immersed in a pre-prepared 12-well “post-soak plate“, containing 2 mL fresh pre-warmed assay medium per well and incubated for 25 ± 2 min at room temperature. Afterwards, the tissues were removed from the assay medium, the medium was decanted off the tissue and the tissues were blotted on a blotting paper. The tissues were transferred to a new 6-well plate (post-treatment plate) containing 1 mL pre-warmed assay medium. The tissues were incubated for 18 ± 0.25 h at 37 +/- 1 °C, 5.0% CO2 / 95% air.
After this incubation period excess medium was removed by blotting bottom on absorbent paper before the inserts were transferred in a pre-prepared 24-well “MTT assay plate” containing 0.3 mL pre-warmed MTT medium and further incubated for 3 h +/- 10 min at 37 +/- 1 °C, 5.0% CO2 / 95% air.
After the 3 h MTT incubation period the inserts were removed, with the bottom of the inserts blotted on blotting paper, and then transferred into new 6-well “extraction plates“, containing 2 mL of isopropanol to extract the formazan only from the bottom of the tissues to avoid possible contamination of test material. The extraction plates were sealed to inhibit isopropanol evaporation. Extraction was carried out after storage overnight in the dark at 2 - 8 °C. At the end of the extraction period the tissues were not pierced to avoid contamination of the extract with remaining test item.
Then the inserts were discarded and the extracts were mixed three times using a pipette. If any visible cell/tissue fragments were in suspension, extracts were centrifuged to eliminate the fragments and avoid further possible interference with the absorbance readings. For each tissue 2 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer using isopropanol as a blank.

- RhCE tissue construct used, including batch number:
EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek), consisting of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.
The EpiOcular™ tissues were provided as kits (e.g. OCL-200-EIT; MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing 24 inserts with EpiOcular™ tissues on agarose (Lot No.: 27017),
1x bottle EpiOcularTM assay medium (Lot No.: 121117ISA),
1x bottle Ca2+/Mg2+-free DPBS buffer (Lot No.: 092817MGKA)

- Doses of test chemical and control substances used:
1. Negative Control: 50 µL Aqua dest. (Sigma, Lot No. RNBF7110)
2. Positive Control: 50 µL methyl acetate (CAS No. 79-20-9 (Lot No.: S6943111)
3. Test Item: 50 mg

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable):
Exposure: 6 ± 0.25 h at 37 ± 1 °C, 5.0% CO2/95% air.
Post exposure post-soak plate: 25 ± 2 min at room temperature
Post exposure post-treatment plate: 18 ± 0.25 h at 37 ± 1 °C, 5.0% CO2/95% air

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
See section "Pre-experiments" in box "Any other information on materials and methods incl. tables"

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2 tissues per group

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm ± 30 nm

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
Mean tissue viability (% negative control) <= 60 %: Irritant (I): UN GHS “Category 1” or “Category 2”
Mean tissue viability (% negative control) > 60%: Non-Irritant (NI): UN GHS “No Category”

Irritation parameter:

other: Relative Tissue Viability (%)

Run / experiment:

Mean of replicates

Value:

44.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The test item showed irritant effects. The mean relative tissue viability (% negative control) was < 60% (44.9%). For detailed information please refer to Table 1 in box "Any other information on results incl. tables".

Interpretation of results:

other: UN GHS Category 1 or Category 2

Conclusions:

In this study under the given conditions the test item showed irritant effects. The test item is classified as “irritant“ in accordance with UN GHS “Category 1” or “Category 2”.

Executive summary:

In the present study the eye irritation potential of N-Benzoyl-5'-O-[bis(4-methoxyphenyl)phenylmethyl]-2'-deoxyadenosine (100% purity) was analysed according to OECD 492 using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium. Hereby, 50 mg of the test item was applied directly atop the EpiOcular™ tissue. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 6-hour exposure and 18-hour post-incubation period and compared to those of the concurrent negative controls. The test item showed no reduction of MTT as compared to the control. Therefore, NSMTT was determined to be 0%.

The test item showed irritant effects. The mean relative tissue viability of two replicates (% negative control) was < 60% (44.9 %). Therefore, the test item is considered to be irritating to the eye in accordance with UN GHS “Category 1” or “Category 2”.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-03-09 to 2018-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted in 2017

Deviations:

no

GLP compliance:

yes

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES:
- Source: Spear Products Inc., 520 Springfield Street, Coopersburg, PA 18036. Fresh eyes were collected from adult cows roughly between the ages of 12-30 months at a slaughterhouse and transported in Hanks Balanced Salt Solution (HBSS) with Ca++ and Mg++ (containing penicillin/streptomycin) on ice to PSL. Corneas were dissected and the assay begun as soon as possible after delivery.

Vehicle:

water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20%

Duration of treatment / exposure:

4 hours at 32 °C

Duration of post- treatment incubation (in vitro):

The optical density at 490 nm was measured upon 90 minutes of incubation with fluorescein after exposure to the test item by using a spectrophotometer.

Number of animals or in vitro replicates:

Three corneas each for the test item, negative control (physiological saline 0.9% NaCl) and positive control (imidazole 20% in physiological saline 0.9% NaCl).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Eyes were carefully examined for defects (opacity, scratches, pigmentation, etc.) shortly after arrival to the facility. Any eyes with defects were omitted from the study and discarded. The tissue surrounding the eye ball was carefully pulled away, and the cornea was excised, leaving a rim of sclera approximately 2 to 3 mm wide. The isolated corneas were maintained in HBSS until all were dissected. Prior to mounting, corneas were again visually examined for blemishes. All corneas free from defects were then mounted in corneal holders (BASF, Duratec) with the endothelial side against the O-ring of the posterior chamber. Each holder was uniquely identified by a matching number on both the anterior and posterior chambers. The anterior chamber was then positioned on top of the cornea and tightened with screws to hold the cornea securely in place and completely seal the chambers. Both chambers were then filled, starting with the posterior chamber to help maintain the normal shape of the cornea, with pre-warmed cMEM, ensuring that no bubbles were formed. The corneas were incubated for a minimum of one hour at ~32 °C to allow them to equilibrate with the medium.

INITIAL OPACITY AND CORNEA SELECTION
At the end of the initial incubation period, the medium in both chambers was replaced with fresh pre-warmed cMEM. An initial opacity (illuminance) measurement (I) was performed on each cornea using an opacitometer (Model: BASF-OP3.0, Duratec). Only corneas having an initial illuminance reading I > Io/1.1651 lux (927 lux) were used for the assay. Io is the empirically determined illuminance through cornea holders containing cMEM without a cornea. Three corneas with an initial opacity closest to the median opacity of all usable corneas were selected as the negative control corneas. Remaining corneas were randomly assigned to test and positive control groups. The medium was removed from the anterior chamber just prior to treatment with test or control substance.

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period, the test or control substance was removed from the anterior chamber and the epithelium was washed at least three times with MEM containing phenol red until no colour change occurred. The corneas were given a final rinse with cMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
Corneal opacity:
The following formula was used to calculate the opacity using the initial and final illuminance measurements taken from each cornea. The values for a and b are equipment-specific variables empirically determined by the manufacturer:
Opacity= (Io/I) - b)/a
Where: a= 0.0251, b= 0.9894, Io= 1080
The value Io is the illuminance through medium without a cornea. This value was previously determined using the mean illuminance through cornea holders containing cMEM. This value is re-evaluated periodically by the testing facility. I is the initial illuminance being evaluated.

Change in Opacity:
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity from the final opacity. The average change in opacity was calculated for the negative control corneas and used to calculate the corrected opacity.
Corrected Opacity:
The corrected opacity value for each cornea treated with test substance or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each treated and positive control cornea. The mean corrected opacity value of each treatment group was then calculated by averaging the corrected opacity values.

Permeability (UV/VIS Spectrophotometer Method):
After the final opacity measurement, the medium was removed from the anterior chamber of the holder. One milliliter of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber of all holders. After addition of the sodium fluorescein solution, all corneas were incubated in a horizontal position for approximately 90 minutes at ~32 °C. Once incubation was complete, the medium from the posterior chamber of each holder was transferred to individual, pre-labeled sample tubes. Aliquots, measuring 200 µL, of each sample were added onto a 96-well plate, in duplicate, with two additional wells filled with cMEM (blanks). The optical density at a wavelength of 490 nm was then measured using a spectrophotometer.
Corrected OD490:
The mean OD490 obtained for the blank wells was calculated. The mean blank OD490 was subtracted from the OD490 of each test or positive control well (corrected OD490).
Final Corrected OD490:
The final-corrected OD490 of the test substance(s) and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea. The mean final corrected OD490 for each treatment group was calculated.
Final-corrected OD490 = corrected OD490 treated cornea – average-corrected negative control OD490

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
Corrected opacity and permeability values calculated above were used for each group to determine the IVIS for each treatment group using the following formula:
IVIS= mean corrected opacity value + (15 x mean corrected OD490 value)

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of triplicates

Value:

7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no prediction can be made

Other effects / acceptance of results:

Proper conduct of the BCOP was confirmed via a positive response with imidazole and a negative response with 0.9% NaCl. For detailed results please refer to table 2 in box "Any other information on results incl. tables".

Table 2: Classification summary

Group	Treatment	Physical State	RESULTS OF LABORATORY TESTING
			Avg Corrected Opacity	Avg Corrected OD	IVIS[1]	Category
						UN GHS	EPA
1	Negative Control (0.9% NaCl)	Liquid	-	-	-	-	-
2	Positive Control (Imidazole)	Powder	100.8	2.508	138	Category 1	I
3	Test Substance	Powder	7.0	0.023	7	No prediction can be made	III

[1] In Vitro Irritancy Score (IVIS)= mean corrected opacity value + (15 x mean corrected OD₄₉₀value)

Interpretation of results:

other: no prediction can be made

Conclusions:

In conclusion, based on the mean in vitro irritation score of 7 obtained in the bovine corneal opacity and permeability assay (BCOP, OECD 437), no prediction can be made regarding the classification of the test substance.

Executive summary:

The eye irritation potential of N-Benzoyl-5'-O-[bis(4-methoxyphenyl)phenylmethyl]-2'-deoxyadenosine (100% purity) was investigated in the bovine corneal opacity and permeability assay (BCOP, OECD 437). The test item was suspended in water to gain a 20% concentration. A mean in vitro irritation score of 7 was determined. The positive and negative controls induced the appropriate responses, indicating the validity of the assay. Based on the results obtained, no prediction can be made regarding the classification of the substance.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The potential of N-Benzoyl-5'-O-[bis(4-methoxyphenyl)phenylmethyl]-2'-deoxyadenosine (100% purity) to induce skin irritation (OECD 439) and eye irritation (OECD 492, OECD 437) was tested in suitable in vitro test methods.

Based on the results, the target substance can be considered non-irritant to the skin. To assess the eye irritant potential of the target substance, the EpiOcular Eye Irritation Test was conducted in accordance to OECD 492. This test is able to correctly identify chemicals that do not require classification or require labelling for eye irritation (Eye Irrit. 2, H319) or serious eye damage (Eye Dam. 1, H318). However, is not possible to distinguish between serious eye irritation and serious eye damage in this test. The test item showed irritant effects in the EpiOcular test. The mean relative tissue viability (% negative control) was ≤ 60% (52.6%). Therefore, the test can be considered to require classification. Subsequently, the test item was tested in the Bovine Corneal Opacity & Permeability (BCOP) assay. The BCOP allows classification as “not classified” and “Eye Dam 1, H318” based on the In Vitro Irritancy Score (IVIS). Results which are in between both categories are identified as “No prediction can be made”. Based on the results from the BCOP test, no prediction can be made regarding the classification of the target substance. Therefore, the test item was considered not to be an ocular corrosive or severe irritant.

By assessing the results from both in vitro eye irritation tests in a weight-of-evidence approach, it can be concluded that classification as Eye Irrit 2, H319 is warranted as it was shown that the substance cannot be considered as non-irritant, but also not as inducing severe eye damage (Eye Dam 1, H318).